Most varieties of crop plants available to agriculture today have been obtained as a result of years of breeding activities focussed on the selection of higher yielding plants adapted to a particular environment. As a consequence, they often lack sufficient genetic variability to adapt to other environments whilst maintaining a high yield. In addition, during their life cycle, plants are exposed to various environmental conditions which greatly influence development and which, when unfavourable, may limit the final yield. Climate and other environmental conditions introduce variability into both total production and in quality of the product obtained over different seasons. Therefore it is a major aim in agriculture to develop varieties with enhanced stability in a quantitative and qualitative sense. Stability in production in the quantitative sense would be beneficial for planning and could avoid anomalies in production. In the qualitative sense, stability would contribute to improve post-harvest treatments and to industrial processing of agricultural products.
Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance are also important factors influencing yield.
The ability to influence one or more of the abovementioned factors, and to thereby increase crop yield, would have many applications in areas such as crop enhancement, plant breeding, production of ornamental plants, arboriculture, horticulture, forestry, production of algae or plants (for example for use as bioreactors, for the production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste or for use as fuel in the case of high-yielding algae and plants).
The final yield of a plant is determined by several parameters amongst which growth is a major contributor. Often an increase in growth correlates with higher yield. Particularly relevant is the capacity of a plant to maintain growth and to continue its developmental programme in unfavourable conditions. Unfavourable conditions are those that limit a plant in achieving its potential maximum production. Given the plant's inability of locomotion as a means of responding to environmental stimuli, plants are exposed to a variety of stresses that limit their performance. Abiotic stress conditions, such as shortage or excess of solar energy, water and nutrients, extremes of hot and cold temperatures, pollution (e.g. heavy-metal pollution) can all have a major impact on plant growth and can significantly reduce plant yield and growth.
The response of a plant to abiotic stresses, such as drought, temperature and osmotic stress, are intimately linked to each other (Zhu et al., Crit. Rev. Plant Sci. 16, 253-277, 1997). Many genes that are regulated by one type of stress are also responsive to the other two. A gene conferring tolerance to, for example, osmotic stress may therefore also confer tolerance to cold and drought stresses. In addition, a plant can be exposed to a multiplicity of stresses during its life cycle e.g. drought stress is often accompanied by high temperature stress. The most common kind of stress plants receive from their surroundings is temperature stress. Each plant species has its own optimal temperature for growth, and its geographical distribution is determined to a major extent by the temperature zone in which it can survive. Recently, concerns have been voiced about the potentially serious effects on agriculture of radical global temperature changes predicted to occur in the near future. There is now an effort to search for practical approaches to improve adaptability of plants to non-optimal temperature conditions. Molecular breeding methods have been applied to address these problems. For example, genetically engineered cold tolerance in plants has been achieved by overexpression of transcription factors such as SCOF-1 or CBF1 (Kim et al., Plant J. 25, 247-259, 2001; Jaglo-Ottosen et al., Science 280, 104-106, 1998); increasing the content of compatible solutes, (Alia et al., Plant Cell Environm. 21, 232-239, 1998); altering membrane lipids; and by reducing the effect of active oxygen species. The ability to withstand high temperatures has been obtained by engineering expression of heat shock proteins, increasing production of compatible solutes, and by altering membrane lipids. However to date there has been no scientific report describing the involvement of plant class-2 non-symbiotic haemoglobin genes in responses to environmental stresses or of plant haemoglobin genes in general in responses to temperature stresses.
Drought, salt stress and high or low temperature stress, are major problems in agriculture because these adverse environmental factors prevent crop plants from maximally exploiting their genetic potential. These stresses influence virtually every aspect of plant physiology and metabolism. Stress generally involves adaptive responses, such as morphological changes in roots or other organs, but also developmental changes, e.g. inhibition of growth. In general the response of a plant can be divided into three categories: maintenance of homeostasis, which includes ion homeostasis and osmotic homeostasis or osmotic adjustment; detoxification of harmful compounds, e.g. of reactive oxygen species or of damaged proteins that originated during the stress; and recovery of growth, that is, relief from growth inhibition and the effects on cell division and expansion imposed during the stress.
Progress has been made through genetic engineering in achieving stress tolerance by manipulating homeostasis, e.g. by increasing the concentrations of osmolytes (Nuccio et al., Curr. Opin. Plant Biol. 2, 128-34, 1999), by overexpressing Na+/H+ antiporters, (Apse and Blumwald, Curr. Opin. Biotechnol. 13, 146-50, 2002) or by overexpressing LEA proteins that may contribute to maintenance of membrane or protein stability (Xu et al., Plant Physiol. 110, 249-257, 1996). Engineering components of the osmotic signalling pathway is also a promising route to achieve osmotic stress tolerance. However, there is no report in the literature establishing a crucial role of haemoglobin genes in improving osmotic stress tolerance.
Haemoglobins are commonly found in a wide range of organisms (Vinogradov et al., Comp. Biochem. Physiol. 106, 1-26, 1993; Bolognesi et al., Prog. Biophys. Mol. Biol. 68, 29-68, 1997). With the possible exception of barley, all examined plant species have at least two haemoglobin genes. These genes have been reported to contain 3 conserved introns, a feature shared with animal haemoglobins (Arredondo-Peter et al., Plant Physiol. 118, 1121-1125, 1998). Based on their structure, plant haemoglobins used to be divided into two groups. The first is a group of symbiotic haemoglobins (leghaemoglobins), comprising haemoglobins that are abundantly present in infected cells of N2-fixing nodules in leguminous plants but that can also be found in non-leguminous plants. The second group comprises non-symbiotic haemoglobins, which are ancestral to the symbiotic type of haemoglobins and which are more widespread in the plant kingdom.
In a more recent classification (Hunt et al., Plant Mol. Biol. 47, 677-692, 2001), haemoglobins were grouped into class 1 or class 2, depending on their amino acid sequence. Haemoglobins that did not fit into either class were assigned to a class 0 that was later renamed into class 3 (Wittenberg et al., J. Biol. Chem. 277: 871-874, 2002). Because the different classes are delineated based on primary amino acid sequences, symbiotic haemoglobins and non-symbiotic haemoglobins may be found in both class 1 or 2. Class 3 comprises the truncated haemoglobins. Members of these three classes not only differ in amino acid sequence, but also in biochemical properties. Truncated haemoglobins are small proteins carrying a haeme group that is able to bind oxygen. Class 1 and class 2 haemoglobins can be discriminated from each other in the conservation of certain amino acids in the sequence (see Hunt et al., 2001 for a detailed description of classes 1 and 2). Class 2 haemoglobins have conserved proline residues at positions B3 and G3, the absence of which may cause a different orientation in the B and G helices in class 1 haemoglobins. Additional substitutions and changes in charge at certain positions in the sequence cause further modifications in the packing of these helices.
The symbiotic haemoglobins are predominantly found in nodules of leguminous and in non-leguminous plants living symbiotically with bacteria. In plants, symbiotic haemoglobins are known to play a role in oxygen transport, thereby stimulating nitrogen fixation by providing oxygen to the nodules. This is made possible by the high affinity for oxygen that the leghaemoglobins have, combined with a fast dissociation constant for oxygen (Appleby, Sci. Prog. 76, 365-398, 1992). Leghaemoglobins belong to a multigene family and are usually posttranslationally modified. The bacterial haemoglobin from Vitreoscilla sp. resembles the leghaemoglobins in its binding properties for oxygen and because of this property the protein has been used to promote growth in plants and micro-organisms (U.S. Pat. No. 5,049,493; U.S. Pat. No. 5,959,187). Vitreoscilla haemoglobin has a KD of 6000 nM, whereas the Arabidopsis class-2 haemoglobin has a KD of 130 nM, which is more than 45 times lower. This high KD makes Vitreoscilla haemoglobin well suited for stimulating oxygen transport and consequently plant growth. Therefore, Vitreoscilla haemoglobin is quite distinct from plant non-symbiotic haemoglobins (Bülow et al., Trends Biotechnol. 17, 21-24, 1999). The use of Vitreoscilla haemoglobin may be compared to the use of bovine haemoglobin for promoting oxygen transfer in plant cell culture media and for plant regeneration (Azhakanandam et al., Enzyme Microb. Technol. 21, 572-577, 1997).
The non-symbiotic haemoglobins on the other hand differ from the leghaemoglobins in their primary protein structure (Arredondo-Peter et al., 1998). In addition, non-symbiotic plant haemoglobins have a very high affinity for oxygen, with a moderate association constant and a very low dissociation constant (about 40 times lower than the dissociation constant for oxygen of symbiotic haemoglobins (Arredondo-Peter et al., 1998; Bülow et al., 1999; Watts et al., Proc. Natl. Acad. Sci USA 98, 10119-10124)). Consequently oxygen is stably bound and a role in oxygen sensing or oxygen transport is not likely (Arredondo-Peter et al., 1998). However little is known about the functions in planta of the non-symbiotic haemoglobins. Their biochemical properties seem to exclude a role in oxygen diffusion, though a role as oxygenase may be possible (Hill, Can. J. Bot. 76, 707-712, 1998). The binding of oxygen causes a conformational change that may affect associated ligand molecules, thereby triggering certain physiological responses (Goodman and Hargrove, J. Biol. Chem. 276, 6834-6839, 2001).
Class 1 haemoglobins are induced by hypoxia, increasing sucrose concentrations (Trevaskis et al., Proc. Natl. Acad. Sci. USA 94, 12230-12234, 1997) or by nitrates (Wang et al., Plant Cell 12, 1491-1510, 2000). They are also expressed in germinating seeds and in roots of mature plants (Hunt et al., 2001) and in differentiating cells (Ross et al., Protoplasma 218, 125-133, 2001). Class 1 non-symbiotic haemoglobins are induced upon hypoxic stress (Hunt et al., 2001). Arabidopsis haemoglobin 1 enhances survival under hypoxic stress and promotes early shoot and root growth in Arabidopsis thaliana (Hunt et al., Proc. Natl. Acad. Sci. USA 99, 17197-17202, 2002). The use of class-1 haemoglobin molecules for altering plant growth characteristics was mainly focused towards manipulating oxygen levels in the plant. Tarczynski and Shen (U.S. Pat. No. 6,372,961) propose the use of maize haemoglobin to modify the oxygen concentrations in a plant cell and to stimulate seed germination and seedling growth of plants. Similarly, overexpression of haemoglobin from barley was shown to increase the ATP content in maize cells and to maintain the energy status under hypoxic stress (Guy et al., WO 00/00597).
The expression pattern of class-2 haemoglobins is different from that of class 1 haemoglobins in that they are expressed during embryogenesis and seed maturation, around openings (e.g. in mesophyl cells of stomata, around the top of the style, around the pore of the nectaries) or at branch points (e.g. to the bolt system, around emerging lateral roots, at the junction of anther and filament) (Hunt et al., 2001). Members of the class-2 haemoglobins are also responsive to cytokinin (Hunt et al., 2001). Harper et al. (WO 02/16655) have shown that haemoglobin 2 is induced in Arabidopsis upon cold, osmotic and saline stress, together with over 400 other genes. However, this class-2 haemoglobin has not been linked to increased stress tolerance. To date, only a few class-2 haemoglobin sequences have been described, among which is the GLB2 from Arabidopsis thaliana and two ESTs from Beta vulgaris that were isolated from stressed seedlings (GenBank acc no BE590299) and from a leaf cDNA library (GenBank acc no BQ586966).